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United States Patent |
5,051,408
|
Cooper
|
September 24, 1991
|
Inulin compositions in gamma polymorphic form
Abstract
A process for preparing gamma inulin comprising the steps of (a)
recrystallizing crude inulin from water at a temperature below 37.degree.
C. to obtain a suspension, (b) heating the suspension at a temperature of
from about 25.degree. to 45.degree. C. for about 1-3 days, (c) further
heating the suspension at a temperature of about 40.degree. to 55.degree.
C. for about 0.5 to 1.5 hours, and (d) isolating insoluble gamma inulin
from the suspension. A composition comprising particles of inulin or an
inulin derivative in the gamma polymorphic form is characterized in that
the particles have a low rate of solution in aqueous media above
30.degree. C., particularly above 37.degree. C. The composition is
effective as the active component of an immunotherapeutic preparation for
activation of the alternative pathway of complement, or for antitumor
treatment.
Inventors:
|
Cooper; Peter Dodd (Monash, AU)
|
Assignee:
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The Australian National University (Acton, AU)
|
Appl. No.:
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501752 |
Filed:
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March 30, 1990 |
Foreign Application Priority Data
Current U.S. Class: |
424/85.1; 424/85.2; 424/85.4; 424/131.1; 424/184.1; 424/278.1; 424/282.1; 514/54; 514/885; 536/4.1; 536/123; 536/123.1; 536/127 |
Intern'l Class: |
A61K 031/715; A61K 039/02; A01N 065/00; C08B 037/18 |
Field of Search: |
536/127,4.1,123,1.1
514/54,885
424/92,93
|
References Cited
Other References
Phelps; Biochem. J. 95:41-47 (1965).
Sommerman et al.; Biochem. Biophys. Res. Commun. 122(1):319-324 (1984).
Cooper et al.; Molecular Immunology 23(8):895-901 (1986).
Cooper et al.; Molecular Immunology 23(8):903-908 (1986).
Cooper et al.; Int. J. Cancer 33:683-687 (1984).
Cooper; Advances in Immunity and Cancer Therapy vol. 1 Ed. Ray; pp. 125-166
Chap. 4 (Sep. 1985).
|
Primary Examiner: Brown; Johnnie R.
Assistant Examiner: Carson; Nancy S.
Attorney, Agent or Firm: Bacon & Thomas
Parent Case Text
This application is a continuation of application Ser. No. 086,634, filed
Aug. 3, 1987, now allowed, and issued as U.S. Pat. No. 4,954,622.
Claims
I claim:
1. A composition comprising particles of inulin, wherein said particles are
in the gamma polymorphic form and are virtually insoluble in aqueous media
at 37.degree. C.
2. A composition according to claim 1, wherein said inulin has a molecular
weight greater than 8,000.
3. A composition according to claim 1, comprising particles of gamma
inulin, wherein the gamma inulin has a molecular weight in the range of
from about 8,000 to about 16,000 and is virtually insoluble in water at
37.degree. C.
4. A composition according to claim 3, wherein said gamma inulin is in the
form of a stable pure suspension of particles <1 .mu.m in diameter.
5. An immunotherapeutic preparation for activation of the alternate pathway
of complement (APC) in a human or animal body, which comprises as the
active component thereof particles of inulin in the gamma polymorphic
form, wherein said particles are virtually insoluble in aqueous media at
37.degree. C.; and a pharmaceutically acceptable diluent or carrier.
6. A preparation according to claim 5, wherein said active component
comprises particles of gamma inulin.
7. A preparation according to claim 5, wherein said carrier or diluent is a
sterile, aqueous vehicle.
8. A preparation according to claim 7, wherein said aqueous vehicle is an
isotonic solution.
9. A preparation according to claim 5 in a form suitable for injection.
10. A preparation according to claim 5 in a form suitable for oral, rectal,
vaginal, topical, nasal or ocular administration.
11. A preparation according to claim 5, further comprising a second active
component which is an immune modulator.
12. A preparation according to claim 4, wherein said immune modulator is a
vaccinating antigen, an antigenic peptide sequence, or an anti-iodiotype
immune globulin.
13. A preparation according to claim 11, wherein said immune modulator is
an interleukin or an interferon or tumor necrosis factor or other
lymphokine, or thymocyte stimulator or other thymus stimulating hormone, a
muramyl peptide or other microbial component or whole microbial component
or whole microbe, or an endotoxin.
14. A method for the activation of the alternative pathway of complement
(APC) in a human or animal body, which comprises administering to the
human or animal body an effective amount for said activation of an
immunotherapeutic preparation according to claim 5.
15. A method for enhancement of an immune response in a human or animal
body to which has been administered an immune modulator which comprises
administering to the human or animal body an effective amount for said
enhancement of an immunotherapeutic preparation according to claim 5.
16. A method for enhancement of the effect of administration of a
vaccinating antigen, an antigenic peptide sequence, or an anti-idiotype
immune globulin, in a human or animal body which comprises administering
as an adjuvant an effective amount for said enhancement of an
immunotherapeutic preparation according to claim 5.
Description
This invention relates to the preparation and identification of individual
polymorphic crystalline forms of inulin, immunotherapeutic preparations
containing inulin and to a method of antitumour treatment by the
administration of those preparations in which inulin is in an insoluble or
particulate form.
"Inulin" is a simple, inert polysaccharide comprising a family of linear
.beta.-D-(2.fwdarw.1) polyfructofuranosyl .alpha.-D-glucoses, in which an
unbranched chain of up to 100 fructose moieties is linked to a single
terminal glucose, the end fructose-glucose pair incidentally being
identical to sucrose; there are no other components. Inulin preparations
are therefore molecularly polydisperse (mol. wts up to 16,000), neutral
polysaccharides of simple, known composition. Inulin is the storage
carbohydrate of Compositae and is cheaply available from dahlia tubers. It
has a relatively hydrophobic, polyoxyethylene-like backbone, and this
unusual structure plus its non-ionised nature allows recrystallisation and
easy preparation in a very pure state.
Although the molecular composition of inulin is well-known, the reported
determinations of its solubility are conflicting. For example, the Merck
Index describes inulin as "slightly soluble in cold water, soluble in
hot", whereas a quantitative study (Biochem. J., 1965, 95, 41-47) suggests
that two distinct forms of inulin exist--the first obtained by
precipitation from water, the second by precipitation from ethanol--both
of which are substantially soluble in water at 37.degree. C. It is also
known that suspensions of inulin become less soluble on standing. The form
obtained from precipitation from water is referred to as alpha-inulin, and
the form obtained by precipitation from ethanol is known as beta-inulin.
However, the conformational differences between the two forms have not
been determined, nor has a method been established which can distinguish
between the various polymorphic forms of inulin.
In developing a method for distinguishing the various forms of inulin, a
third, hitherto unknown, polymorph has been discovered and isolated.
This third polymorph, hereinafter referred to as gamma-inulin, is virtually
insoluble in water at 37.degree. C., but is soluble at temperatures in the
range of 70.degree.-80.degree. C., as are the alpha and beta forms. The
series of polymorphic forms in which inulin crystallises may be
characterised by their different solubility rates in aqueous media ranging
from one instantly soluble at 23.degree. C. (beta .sub.23.sup.0 inulin)
through a form soluble at 37.degree. C. with a half-time of 8 minutes
(alpha .sub.37.sup.8 inulin) to a form virtually insoluble at 37.degree.
C. (gamma inulin). All forms are interconvertible, the more soluble and
unstable progressing on standing to less soluble and more stable forms,
only reversible by complete solution followed by recystallisation. The end
product is the stable gamma inulin.
According to the present invention, there is provided, a composition
comprising particles of inulin or an inulin derivative in the gamma
polymorphic form, characterised in that said particles have a low rate of
solution in aqueous media above 30.degree. C.
The active components which may be used in accordance with this invention
include not only inulin, .beta.-D-[2-1]-polyfructofuranosyl
.alpha.-D-glucose, but also derivatives thereof including .beta.-D-[2-1]
polyfructose which may be obtained by enzymatic removal of the end glucose
from inulin, for example using an invertase or inulase enzyme capable of
removing this end glucose. Other derivatives included within the ambit of
this invention are derivatives of inulin in which the free hydroxyl groups
have been etherified or esterified, for example by chemical substitution
with alkyl, aryl or acyl groups by known methods.
The active component is preferably of molecular weight greater than about
3000, more preferably, greater than about 8000.
According to one preferred aspect of the present invention, the composition
comprises particles of gamma inulin, which is characterised in that:
(a) its molecular weight is in the range of from about 8,000 to about
16,000; and
(b) it is virtually insoluble in water at 37.degree. C.
In one particularly preferred aspect, this invention provides a composition
comprising gamma inulin as described above in a stable, very pure
suspension of particles <1 .mu.m in diameter. Such a suspension has been
found to be a reagent specific for in vivo and in vitro activation of the
alternative pathway of complement (APC) as described in detail below.
It is envisaged that those inulins or inulin derivatives whose molecular
weights are too low to convert to the gamma form may be rendered less
soluble, and therefore easier to prepare in insoluble crystals, by
appropriate chemical substitution, for example by substitution with alkyl,
aryl or acyl groupings.
The present invention also provides a process for the preparation of gamma
inulin from any convenient source such as commercially available inulin.
Broadly speaking, the process comprises the steps of:
1. removing trace impurities;
2. recrystallising from water (preferably at alkaline pH) at a temperature
well below 37.degree. C. to obtain a finely divided particulate in
suspension;
3. heating said suspension at a temperature in the range of from about
25.degree. to 45.degree. C. for approximately 1 to 3 days;
4. further heating said suspension at a temperature in the range of from
about 40.degree. to 55.degree. C. for approximately 0.5 to 1.5 hours; and
5. isolating the thus-formed insoluble gamma inulin from the suspension.
The following steps comprise a purification procedure for the production of
endotoxin(ET)-free "gamma inulin for injection":
1. Inulin powder (obtained, for example, from commercial sources) is first
treated to remove trace impurities. This is achieved by washing by
suspension in water and resedimentation, recrystallisation from water with
minimal heat (preferably less than 70.degree. C.) and cooling or
freeze/thawing, and passing in the dissolved state through ion-exchangers
such as DEAE-cellulose and sulphonated polystyrene resin. The pH is
preferably kept above 6.5. The solution is then sterilised and freedom
from endotoxin completed by filtration through an appropriate filter (for
example, Zetapor 0.2 .mu.m SP grade charge-modified nylon 66 membrane).
2. The solution is converted to a finely divided precipitate (particles
preferably less than 1 .mu.m diameter) of a soluble inulin (mainly alpha
form) by recrystallisation at a temperature well below 37.degree. C.,
preferably 5.degree. C., and preferably at a high pH (for example, by
using a 0.1% ammonia solution) and a concentration preferably greater than
50 mg/ml inulin. After several days, usually 5-7 days, most of the inulin
has crystallised.
3. The suspension is then heated to a higher temperature, preferably in the
range from 30.degree. to 40.degree. C. for a further period of
approximately 1-3 days, when most of the inulin precipitate has converted
to the gamma form.
4. The suspension is then further heated for a shorter period of
approximately 1 hr at a higher temperature, preferably in the range from
45.degree. to 50.degree. C., to complete the conversion and to dissolve
any alpha inulin incapable of conversion.
5. The suspension may be largely freed of soluble matter by sedimentation
and resuspension in water.
The suspension is then resuspended to a standard concentration, for
example, 62.5 mg solid inulin/ml, which yields 50 mg/ml isotonic saline
when mixed with one-quarter vol. 4% w/v saline solution. Concentrations
may be measured with a refractometer. The degree of dispersion is checked
by appropriate procedures, for example, density gradient centrifugation or
electron microscopy.
At all steps after stage 1 described above, endotoxin-free materials and
full aseptic techniques are employed. The suspension may be adjusted to
isotonicity with saline solution before or after standing at 5.degree. C.
Apart from some slight hydrolysis, it is stable up to 45.degree. C. but is
preferably not heated further. After freezing and thawing the suspension
is still active but the particles may become aggregated.
Earlier research suggests that there exists some fundamental immune
principle potentially exploitable for cancer therapy. Unfortunately, the
role of the immune system in the genesis/elimination of cancer is not
fully understood and remains an area of considerable controversy and
doubt. There are many factors involved and the current understanding of
the mechanism of action of the immune system on tumours at the cellular
level is poor.
From a study of virtually all of the known immuno-potentiating agents
(approximately 20 compounds) with well substantiated antitumour activity
(Cooper, P. D., Advances in Immunology and Cancer Therapy, Vol.1, Chapt.4,
pp125-166 (1985), Ray, P. K. (Ed.), Springer-Verlag, N.Y.), it has been
shown that they either activate the alternative pathway of complement, or
they activate macrophages (apparently through their endogenous APC), or
both. Thus, despite the great chemical diversity in the molecular
structure of these compounds exhibiting antitumour activity, APC
activation appears to be a common property. Further, it has been shown
that two purified agents which are highly specific for APC activation
(i.e., where cytotoxicity and other factors could be ruled out) have
significant anti-tumour activity in carefully controlled experiments with
specific strains of mice. These two agents were isolated complement
component C3b and isolated cobra venom factor (Cooper, P. D. and Sim, R.
B., Int.J.Cancer, 33, 683-687 (1984)). From this earlier research, it was
concluded that other specific APC activators might also be expected to
show antitumour activity.
Accordingly, an object of the present invention is the provision of a
preparation which, when administered to a patient suffering from cancer,
will affect the alternative pathway of complement to reproducibly and
significantly increase the survival time, or improve the quality of life,
of that patient.
Another object is the provision of a preparation which, when administered
to ostensibly healthy persons at intervals during their lifetime, will
eliminate altered cells in their precancerous stages and reduce the chance
of overt cancers appearing later in life.
It has been established that inulin, when administered in its insoluble or
particulate form, especially in the gamma polymorphic form as described
above, is a potent APC activator and has a significant antitumour effect
in mice.
Standards for (dissolved) "inulin for injection" are given in the British
and U.S. Pharmacopoeias. Dissolved inulin and probably its hydrolysis
products are excreted with a half-time of one hour in humans; the ultimate
products of hydrolysis (fructose and glucose) are simple food-stuffs. The
British Pharmaceutical Codex (1979) states that the only pharmacological
effect of (dissolved) inulin is an osmotic diuresis at higher doses. Thus
the only effect of particulate inulin, if any, should be related to its
physical state. No nephrotoxicity or antigenic effects have been found in
rabbits given a massive i.v. course of particulate inulin, and the only
reported immunological interaction besides APC activation has been some
cross-reaction with certain myeloma proteins derived from previously
experienced bacterial levans. It should be noted that no attempts appear
to have been made to purify or to define the physical forms of the inulin
used.
In work leading to the present invention, it has been found that high
purity inulin, when administered in its insoluble or particulate form,
even at low dose rates, is a potent activator of the APC in vitro in mouse
or human serum, the order of activity being similar to the most powerful
activators known. The classical pathway of complement is unaffected. It
has also been found that insoluble or particulate inulin has a potent
anti-tumour effect on B-16 melanoma cells when given i.p. to C57 black
mice, which display a 55% increase in mean survival time.
Accordingly, the present invention provides in a further aspect an
immunotherapeutic preparation for activation of the alternative pathway of
complement, or for antitumour treatment, which comprises as the active
component thereof, particles of inulin or an inulin derivative in the
gamma polymorphic form, characterised in that said active component has a
low rate of solution in aqueous media above 30.degree. C.
In a further aspect, the present invention extends to the use of an
immunotherapeutic composition a broadly outlined above for the activation
of the alternative pathway of complement in the human or animal body, or
for antitumour treatment in the human or animal body.
Preferably, the active component is gamma inulin as characterised above.
The administration of an immunotherapeutic preparation as described above
may be performed by any convenient means, for example by intraperitoneal,
subcutaneous, intravenous or intra-tumour injection.
Of the three polymorphic forms of inulin now known--alpha, beta and
gamma--it is preferable, in the present invention, to employ substantially
pure gamma inulin having a molecular weight of at least 8000--more
preferably 9000 to 12000--and formulated in an injectable preparation. The
gamma form is preferred because it has been found that the ability of
inulin to activate the APC and its anti-tumour effect is correlated with
its insolubility, and the gamma form is the most insoluble and thus the
most active. Further, dissolved inulin and polymorphic forms capable of
dissolving substantially at 37.degree. C. can interfere with these
activities, and accordingly it is preferred that the suspension of inulin
be free of the alpha and beta polymeric forms.
Gamma inulin for injection is preferably formulated as a sterile, milky
suspension comprising 30-60 mg, preferably 50 mg, of pure, insoluble
inulin particles per ml of saline, largely free of dissolved inulin and of
endotoxin (less than 0.1 ng/ml by limulus amoebocyte lysate assay). Such a
preparation has a significant but very low intrinsic pyrogenic effect, but
passes the British Pharmacopoeia (1980) test for pyrogens at a dose of 10
mg/kg. It is expected to be free from traces of protein, lipid, nucleic
acid and charged polysaccharides, and from soluble materials other than
inulin or inulin hydrolysis products. The suspension is stable in the
temperature range of, for example, from 0.degree. C. to +45.degree. C. and
is preferably stored at 2.degree.-8.degree. C. as an aqueous suspension
from which it slowly settles. A preservative such as phenyl mercuric
nitrate (British Pharmacopoeia, 1980), for example at 20 .mu.g/ml, may be
included in the suspension without loss of in vivo or in vitro activity.
The suspension is easy to handle and to inject, but must not be frozen or
heated over 45.degree. C. The particles may be prepared as ovoids less
than 1 micron in diameter and do not tend to aggregate, and thus should
not block microvasculature.
Further details of the preparation and use of the active components of the
present invention are given in the following Example. In this description,
all temperatures are in degrees Centigrade, and technical terms and
abbreviations have the usual meaning in the art. Crude reagents, products
and preparations can be purified by the means described herein, or by
other means known in the art.
EXAMPLE 1
Inulin preparations
a Inulin (90% w/w, Sigma, St.Louis, Miss., from dahlia tubers) was
processed aseptically; final solutions were sterilized by membrane
filtration. Dry weight was by refractive index at 26.degree. C. All
air-dried preparations contained .about.10% H.sub.2 O (w/w) and were
polymers of fructose free of monomer [by reaction with Fehling's solution
and mobility in ascending paper chromatography after but not before brief
boiling in 2M trifluoroacetic acid, using the AgNO.sub.3 (Trevelyan W. E.,
et.al., (1950) Nature, Lond. 166, pp.444-445) and resorcinol (Phelps, C. F.
(1965) Biochem. J., 95, pp.41-47) sprays]. Major and minor contents of
fructose and glucose, respectively, were confirmed in all preparations
using chromatography in chloroform:acetic acid:H.sub.2 O (6:6:1) and
water-saturated phenol after hydrolysis. Other reducing sugars were
undetectable (<1%). All preparations were free of N and S, and C and H
contents and specific rotations were as expected.
b. Inulin I. Crude inulin was stirred and washed twice at 23.degree. C.
with 0.1% (v/v) ammonia in deionized water (40 ml/g inulin); .about.80%
was insoluble.
c. Inulin II. Inulin I, dissolved in 0.1% ammonia (5 ml/g inulin,
69.degree. C.) was slurried with 1% (w/w) DEAE cellulose (Eastman Kodak,
Rochester, N.Y.) prepared for chromatography (Himmelhoch, S. R. (1971) in
Methods in Enzymology, (Edited by Jakoby, W. B.), Vol.22, pp.273, Academic
Press, New York), and the frozen filtrate was then allowed to stand
(23.degree. C., 48 hr), the filtered cake washed (5.degree. C., 0.1%
ammonia then dry acetone) and air-dried (yield 65-70%). Inulin II was the
starting point for preparations described herein. Phenol-water
chromatography after hydrolysis allowed elution and assay by the
phenol-sulphuric method (Dubois, M. et. al. (1956), Analyt. Chem., 28,
350-356), giving a fructose:glucose ratio of 20-80:1, consistent with a
glucose terminal on the mol.wt. range described. Ash, P and O.D. (260 nm)
were absent (crude inulin contained 0.6% w/w, 0.08% w/w and trace,
respectively). Acetone washes contained no lipid. Titration of inulin II
(10% w/v, pH 6.7, 20.degree. C.) with N/100 HCl failed to detect carboxyl
groups. Zero ash, and N after crystallisation from 0.1% ammonia, indicated
zero anionic groups, and with zero S and P showed that ionizable
contaminants were undetectable. O.D. scans (62.5 mg/ml) showed no peaks
from 700-240 nm, provided that caramelization was avoided. The particles
from aqueous crystallisation are smaller with more rapid crystallisation,
as induced at higher concentration and pH, lower temperature and ionic
strength, and by colloidal seeding.
d. ET-free gamma inulin for injection. Batches of different original alpha,
beta and gamma content can be used, provided that sufficient molecules are
>8000 mol.wt. Hardware should be pre-soaked in alkaline detergent (Decon
90, 5% v/v; Selby, Sydney); glassware should also be baked (3 hr,
195.degree. C.) then assembled with oven-labile materials and autoclaved
(Weinberg, 1981). Water is deionized by Milli-RO Reverse Osmosis then
"polished" by Milli-Q filtration (Millipore, Sydney) and is then ET-free
(Limulus assay). Water or solutions should be additionally treated to
remove ET by filtration through sterile 0.2 .mu.m Zetapor SP grade
charge-modified nylon 66 membranes (AMF Cuno, Meriden, Conn.), and then
autoclaved.
Crude inulin was dissolved by stirring (40 g, 800 ml 0.1% ammonia,
75.degree. C.), clarified hot (Whatman No.42 paper), frozen (-15.degree.
C.) and then allowed to stand with 1 ml CHCl.sub.3 (37.degree. C., 3-4
days). The precipitate was washed twice (800 ml water, 23.degree. C.),
dissolved in recently boiled water (pH 6.5-7, 250 ml, 75.degree. C.), and
filtered slowly at 5-6% w/v and <40.degree. C. through a 7 cm diameter, 2
cm deep bed of washed DEAE-cellulose. The filtrate (made 0.1% ammonia and
70.degree. C.) was filtered through a similar bed of Amberlite sulphonic
acid resin (CG-120, BDH, Poole, adjusted to the ammonium form), reheated
with more ammonia (0.1%, 70.degree. C.) then filtered (Zetapor).
The sterile, ET-free solution (pH 9-10, 5% w/v) was stirred (5.degree. C.,
5-7 days); if too large, the crystals should be redissolved with more
ammonia (to 0.2%) and briefly heated to faint turbidity, which then
provides seeding nuclei, then re-stirred (5.degree. C., 3-5 days then
37.degree. C., 2-4 days). The particles (90-99% gamma inulin) were
centrifuged (30 min, 4000 g), resuspended by shaking (400 ml water,
50.degree. C.) then heated (1 hr, 50.degree. C.) and washed twice
(5.degree. C.). Yields are 40-50% but depend on mol.wt. of the starting
material. The final suspension at 62.5 mg/ml was checked for dissolved
matter (<0.2% w/v), particle size (see below), insolubility (<10% drop in
O.D. at <1 mg/ml, 37.degree. C., 24 hr), ET content (Limulus assay,
"E-toxate", Sigma) and sterility at 23.degree. and 37.degree. C. It was
stored at 2.degree.-8.degree. C. (although nothing dissolves after 18 days
at 37.degree. C., by refractive index); it must not be frozen or heated
above 45.degree. C. No aggregation will occur if unfrozen. The undiluted
suspension settled very little and resuspended easily. Gamma inulin for
injection is a sterile, ET-free (<6 pg ET/mg) suspension of 50 mg finely
dispersed, insoluble gamma inulin per ml of 0.8% NaCl containing 20
.mu.g/ml phenyl-mercuric nitrate, PMN [Fluka, Buchs, Switzerland,
recrystallised (British Pharmacopoeia, 1980) and Zetapor-filtered] to
facilitate multiple entry. Gamma inulin (50 mg/ml) had no effect on the
antibacterial action of PMN. The suspension is stable for at least 22
months at 5.degree. C.
e. Characterisation of gamma inulin. Electron micrographs (phosphotungstate
stain) of gamma inulin for injection revealed ovoids of 0.7-1.4 .mu.m
diameter.
Molecular weight determinations were performed on gel chromatography
columns on PBS. Calibration of a Biogel P-30 column with standard
polysaccharide markers showed a peak of 8,500-10,000 (median.about.9300)
mol.wt. for gamma inulin, equivalent to 52-65 hexoses. Gamma inulin on a
Sephadex G-50 column had a mol.wt. of .about.300.
f. Solubility forms of inulin. Difficulties arising from the instability of
the polymorphic forms of inulin were resolved by following decreases in
turbidity (O.D., 700 nm) of <1 mg/ml finely divided inulin suspensions in
cuvettes at 37.degree. C., as some measure of their solution rate in vivo.
They were resuspended by pipetting, then stood briefly before reading in
the spectrophotometer. The turbidity curves of different preparations were
then found to change in a consistent manner as shown in FIG. 1.
The turbidity changes were reproducible and clear-cut and provided a
convenient monitor to identify at least the following solubility forms
(for "alpha" and "beta" nomenclature, see McDonald, E. J., (1946), Adv.
Carbohyd. Chem., 2, 253-277).
1. beta .sub.23.sup.0 inulin (very rapidly soluble at 23.degree. C.);
2. beta .sub.37.sup.0 inulin (soluble slowly at 23.degree. C. but very
rapidly at 37.degree. C.);
3, 4 and 5. alpha .sub.37.sup.2, alpha .sub.37.sup.8 and alpha
.sub.37.sup.15 inulin (respectively soluble with half-times to reaching a
plateau of turbidity of 2-4, 8 and 15 min. at 37.degree. C.);
6. gamma inulin (slightly or undetectably soluble at 37.degree. C.).
g. Toxicity of gamma inulin. A group of 5 mice accepted a course of 3 doses
of 25 mg of purified gamma inulin over a period of 9 days without evident
distress (total dosage 2.5 g/kg), but on sacrifice at day 10 their livers
and spleens were found to be enlarged. Intravenous administration at 25
mg/kg caused collapse of the animals in 15-20 min followed by complete
recovery, and the LD.sub.50 by this route in mice was about 100 mg/kg.
EXAMPLE 2
a. Assays for APC activation
The assays depend on the known ability of the APC in mouse or human serum
diluted in EGTA/Mg.sup.2+ buffer to specifically lyse rabbit red blood
cells (RBC). Activation of the APC develops labile reactive intermediates
which rapidly decay. APC `activators` thus destroy the APC in the serum,
which then becomes unable to lyse rabbit RBC added subsequently. The
amount of lost lytic activity is a measure of the amount of activation.
Full details of these assays are ser out in Cooper, P. D. and Carter, M.
(1986) Molecular Immunology, 23, 8, 895-901 and 903-908, incorporated
herein by reference.
1. In vitro activation
Standardised portions of a serum diluted in EGTA/Mg.sup.2+ buffer are
incubated for 30 min. at 37.degree. with graded doses of an activator
suspension. The activator is then removed by centrifugation and standard
portions of the supernatant are reincubated at 37.degree. for a standard
time with a known number of washed rabbit RBC. The amount of activation is
measured as the proportion of unlysed cells quantitated by optical density
at 640 nm.
2. In vivo activation
Graded doses of gamma inulin are inoculated i.p. into groups of three or
four mice per time point, and the mice killed after various times, blood
collected and serum pooled. Care is taken, using known procedures, to
conserve complement activity. Standard portions of a series of dilutions
in EGTA/Mg.sup.2+ buffer of these sera, in comparison with similar
dilutions of a contemporary pooled serum from untreated mice of the same
batch, are incubated at 37.degree. for a standard time with a calibrated
number of washed rabbit RBCs. The amount of activation is measured as the
proportion of unlysed cells quantitated by optical density at 640 nm.
b. Antitumour activity. Full details of antitumour activity of gamma.inulin
in mice are set out in Cooper, P. D. and Carter, M. (1986) Molecular
Immunology, 23 8, 903-908, incorporated herein by reference.
Mouse melanoma cells were grown in DMEM medium supplemented with 5% foetal
bovine serum (both from Gibco, Paisley, Scotland). Cultures were split 1:8
every 7 days, yielding 1-1.5.times.10.sup.7 cells per 75-cm.sup.2 flask,
and the mice were inoculated i.p. with 1.times.10.sup.6 freshly harvested
cells in 0.2 ml PBS, as previously described (Cooper, P. D. and Masinello,
G. R., Int.J.Cancer, 32, 737-744 (1983)). All preparations were
administered i.p., and there was no sign of distress in the animals after
any dose. The survival times of individual mice were recorded daily and
the significance of differences in mean survival time calculated by
Student's t-test. All the mice were sex- and age-matched C57BL/6J; test
samples comprised seven mice each and controls 14 or 21 mice each.
RESULTS
a. APC Activation
It was found that gamma inulin is a potent activator of the APC in vitro in
mouse or human serum at 2-8 .mu.g/ml. This is an order of activity similar
to the most powerful activators known.
FIG. 2a, left, compares in simultaneous tests the in vitro activation of
the APC in human serum by gamma inulin with two other well-known
activators, zymosan and SAC (Staphylococcus aureus Cowan type I, heat
killed suspension). In these tests, a high unlysed RBC value indicates a
high degree of APC activation in the serum.
FIG. 2b, right, compares the APC activation ability in human serum of the
several forms of inulin, using the s=me method. Mouse serum gave almost
identical results.
When given i.p. in mice, a minimum dose of 50 .mu.g inulin (2.5 mg/Kg)
produced detectable in vivo APC activation in the serum when serum is
collected 2-16 hr after injection of the inulin (FIGS. 3a-g). This is a
concentration comparable to the minimum dose for in vitro APC activation
by inulin (FIG. 2).
The classical pathway of complement was unaffected.
b. Anti-tumour activity
FIG. 4 depicts curves illustrating the increased survival time after gamma
inulin treatment of C57 black mice inoculated i.p. on day 0 with B16
melanoma cells. The mice were given 15 .mu.g of gamma inulin i.p. on day 1
and again on day 4 post-inoculation (p.i.)
DISCUSSION
It has been shown that gamma inulin has a potent anti-tumour effect on B16
melanoma cells. Treated mice displayed a 55% increase in mean survival
time with doses as low as 1 mg/kg. Thus the lowest doses giving APC
activation in vitro and in vivo are closely correlated with the minimal
effective anti-tumour dose. The effect is not lost at doses as high as 5
mg/mouse (250 mg/kg). The more rapidly soluble forms of inulin (alpha and
beta) are almost as active, but in these cases the action is blocked at
higher doses. When inulin is dissolved by brief heating at
60.degree.-70.degree. its in vitro APC activation and its in vivo
anti-tumour effect are simultaneously lost, but are regained on
recrystallisation at lower temperatures. This effect, and its low dose
requirement together with its high purity, confirms that it is the
particulate inulin which is the active constituent.
From what is currently known of non-specific active immunotherapy, the
optimal administration of preparations of the present invention is likely
to be as close to the tumour as possible, i.e. the intratumour plus
intravenous routes, representing a combination of regional and systemic
treatment. Intracavity routes (peritoneal or thoracic) are expected to be
useful, especially for effusions, and translated within a few hours into
an activation of the APC in the blood. Intramuscular, subcutaneous and
intradermal inoculations are likely to have a moderate "depot" effect and
will be particularly useful to treat affected lymph nodes as the gamma
inulin will tend to drain into them. Granuloma formation has not been seen
in mice, cats or dogs by these routes. Orally, inulin will probably be
digested but may be usefully passed to the intestinal mucosa by a
delayed-release formulation. Topical application is expected to be
effective. Sensitive tumour types are unknown, but those directly
accessible to contact with inulin, e.g. in the blood, or with good blood
supply, should be more susceptible. It is expected that the patient should
be immunocompetent, and thus its most likely use as an adjunctive treatment
will have to be carefully evaluated in terms of the cytotoxic or
irradiation regimens included. Before timing and dosage can be determined,
the maximum safe degree of activation of the APC in humans has to be
ascertained. Such activation will probably have to be repeated as
frequently as is allowed by the natural regeneration of the alternative
pathway, which probably returns to normal levels in 24-48 hr. Initially,
it is suggested that the following dose regimens might be effective--5-50
mg per adult human every 14 days, about half i.v. or s.c. and half
intratumour (for comparison, the usual dose of dissolved inulin is 3 g
i.v. initially, plus 7 g over the next few hours). It is likely, from
rates of APC activation tolerated by renal dialysis patients, that the
total dose of gamma inulin to be given intravenously will be given quite
slowly, say 5 mg/10 minutes, perhaps by infusion in diluted form or by a
micropump. The degree of APC activation should be monitored carefully by
in vitro tests, and the dose of inulin increased until detectable but
subtoxic APC activation is observed. It is desirable also to follow a
variety of other immunological parameters, such as macrophage, T and B
cell activation, and natural killer cell activity.
The main side effect to be expected in humans is from direct, acute
activation of the alternative pathway, mainly via the anaphylatoxins C3a
and C5a. Mice happen to be very resistant to shock from this source. If
C5a exceeds a certain blood level in humans the outcome appears to be
irreversible, and there are other undesirable effects, e.g. from
granulocyte emboli. One study with dissolved inulin in humans showed that
10-14 per cent APC activation passed without clinical remark, but another
showed in haemodialysis patients that production by the dialysis membranes
of greater than 8.5 .mu.g/ml of the activation product C3a desArg produced
undesirable clinical symptoms.
It is envisaged that gamma inulin may be usefully applied to internal or
external body surfaces, where the inulin particles may pass into the
body's circulation. Alternatively the gamma inulin, present in an external
wound or on internal moist surfaces, may activate leukocytes which are then
likely to migrate into the body to exert their immune influence. For these
purposes gamma inulin may be applied topically to the skin or, in a
suitable delayed-release vehicle, pass through the stomach to be liberated
on intestinal mucosa or, in other vehicles such as suppositories, drops or
aerosols, be inserted into the rectum, vagina, nose, throat, eyes or upper
and lower respiratory tracts.
Gamma inulin is stable at room temperature and may be supplied in the usual
pharamceutically acceptable formulations, vehicles and preparations, namely
dried powders or suspensions in distilled water, saline or isotonic
solutions, with or without preservatives. It may easily be sterilised by
filtration and made into an injectable preparation free from endotoxin.
Gamma inulin, being inexpensively available, is as suitable for veterinary
as for human applications.
The action of gamma inulin represents a single, clean signal to the immune
system, namely the activation of the alternative pathway of complement
(APC). The purity of this signal is important in allowing the elimination
of undesirable side effects. However, the immune system is extremely
complex and usually responds in nature to a number of different immune
signals from stimulating entities (for example, a noxious microbe or
parasite, a cancer cell, a vaccinating antigen or an allergenic substance,
among others). It is by the interaction of such signals that the body
achieves its powerful response to a very large variety of "foreign"
stimuli. It is therefore natural to expect that gamma inulin will achieve
its most potent effect in synergistic action with other immune-stimulating
signals.
An important application for gamma inulin is as an enhancer or immune
adjuvant to a vaccinating antigen or to substances that immunologically
mimic the three-dimensional structure of the antigen's reactive region
(its epitope). These substances are often poorly antigenic on their own.
They may comprise carefully designed peptide sequences or may be
antiidiotype immuno globulins. The latter have as their eliciting antigen
the idiotype (region binding to an epitope) of those immune globulins
elicited by the original antigen. The antiidiotype, by being complementary
to a structure itself complementary to the original antigen, thus resembles
that antigen in its three-dimensional structure.
Gamma inulin has been found to have vaccine adjuvant activity. As an
example of this, an antigen (either bovine serum albumin or keyhole limpet
haemocyanin) inoculated into mice elicited substantially more antibody if
given as a mixture with gamma inulin than if given on its own. Groups of
mice were injected with each preparation and the mean antibody
concentrations measured in micrograms/ml by radioimmune or ELISA assays.
In one test, the antibody concentration elicited by the mixture of gamma
inulin and antigen was 6.2 times that elicited by the antigen alone
(p<0.001), while the antibody elicited by the antigen in an emulsion with
Freund's Complete Adjuvant (a known, powerful adjuvant that is too toxic
for human application) was 10.4 times that of the antigen alone (p<0.001).
Other immune modulators that are likely to act synergistically with gamma
inulin are:
a. the interleukins, the interferons, the tumour necrosis factors and many
other identified immune stimulatory factors that are collectively known as
lymphokines or cytokines;
b. thymocyte stimulators such as levamisole, or the several thymus
stimulating hormones, one of which is thymosin;
c. macrophage stimulators such as the muramyl peptides or other microbial
components;
d. endotoxin;
e. whole microbes.
By way of example of such synergism, it was found that a mixture of crude
interferon and crude tumour necrosis factor, injected with gamma inulin
into mice previously inoculated with the B16 melanoma, gave a mean
survival time >30% greater than either gamma inulin or the lymphokine
mixture on their own, neither of which produced any survivors. More
importantly, gamma inulin plus the lymphokines eliminated the tumour
entirely from ca. 30% of the mice, a finding rarely made in this system.
Similar results are obtained with the thymocyte stimulator succinyl
concanavalin A.
An immune stimulator such as gamma inulin is likely to have a beneficial
effect on any human or animal disorder with an immunological component.
Cancer cells can be recognised by the immune system as foreign to the
body, or "nonself". The beneficial effect of gamma inulin on cancer in a
mouse model has been demonstrated above, and this benefit, with or without
synergistic action of other immune modulators, is likely to extend to human
or animal cancer patients.
An extension of this benefit is as follows. The carcinogenic process,
whereby a normal cell is changed to a fully malignant cell, is known to
occupy many years in man. During this process, of which the eventual
victim is usually unaware, the damaged cell multiplies slowly and passes
through several stages in which its progeny are not yet fully malignant
but nevertheless may be recognised by the immune system as abnormal or
nonself. This would require a boosting immune stimulus in those cases
otherwise destined to escape detection by the body's immune defences.
Consequently a treatment during the carcinogenic process with an immune
stimulator with negligible side effects, such as gamma inulin, is likely
to eliminate the premalignant cells and lessen the chance of later
emergence of fully malignant progeny cells. Thus regular treatment with
gamma inulin, with or without other immune modulators and at say three
year intervals, of persons at risk (for example those over 40 years of age
and/or those with identifiable high risk factors) is likely to decrease the
overall incidence of malignant disease in the community.
Infections with microbes, worms or parasites, particularly those of a more
chronic course, are likely to be combatted by appropriate treatment with
gamma inulin, with or without other immune modulators. Other immune
disorders such as allergic or rheumatic diseases, immune deficiency
diseases, or neurological or gastro-intestinal disorders related to
dysfunction of the immune system, ar likely to be similarly responsive.
Those skilled in the art will appreciate that modifications and variations
to the invention described above are possible without departing from the
present inventive concept.
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